The overarching aim of this application is to elucidate the principles that govern enzyme catalyzed C-H and O2 activation processes. Studies of C-H activation (proton, hydride and hydrogen atom) are focused on understanding the role of quantum mechanical hydrogen transfer and its link to protein structure and dynamics. The investigation of oxygen activation is directed toward reactions that remain particularly enigmatic with regard to the nature of the oxygen species and/or the reaction pathway that leads to product formation. The pharmacologically important enzyme lipoxygenase has become a center pin of studies aimed at understanding the role of hydrogen tunneling in enzyme catalysis;future studies include probes of protein motions and their links to the tunneling event, the impact of high pressure on tunneling, and the characterization of a thermophilic homolog for comparison to the mesophilic soybean lipoxygenase. The channeling of oxygen within a discrete region of protein, orthogonal to the substrate-binding pocket, has been postulated in lipoxygenase;this will be further tested using site-specific mutagenesis. Tyramine ?- monooxygenase (T2M) is a member of a small family of eukaryotic copper proteins that play essential roles in the generation of hormones/neurotransmitters;a detailed program is described that examines key regions of the protein in relation to the C-H and oxygen activation processes. The copper enzyme galactose oxidase (GalO), postulated to generate a similar Cu(II)-superoxo- intermediate, will be contrasted to T2M as a benchmark for understanding O-H vs. C-H activation. The 2-His, 1-Asp family of non-heme enzymes utilizes a common active site to functionalize a wide range of disparate reactions. Studies are described to examine two members of this family that depend on different reductants;ascorbate in the 1-aminocyclopropane 1- carboxylate oxidase (ACCO) reaction and alpha-ketoglutarate in the taurine dioxygenase (TauD) reaction. TauD is targeted for detailed studies of tunneling, while studies of ACCO will focus on the enigmatic pathway for ACC breakdown and its link to oxygen activation. Finally, two enzyme systems that catalyze removal of a charged hydrogen species as a proton, catalyzed by copper amine oxidase (CAO), and as a hydride ion, catalyzed by glucose oxidase (GO), provide unique opportunities to interrogate the inter- relationship between substrate specificity (CAO) and reaction driving force (GO) in the tunneling process. The above studies are of fundamental significance to our understanding of Nature's catalysts, with previously published findings from this laboratory leading to paradigm shifts regarding the physical origins of enzyme catalysis. Understanding the latter provides guidelines for the synthesis of small biomimetic catalysts, for the design of novel protein/peptide based catalysts and for the establishment of conceptual platforms that guide drug design. PUBLIC HEALTH RELEVANCE Two of the most fundamental processes that underlie aerobic life are C-H and O2 activation. The goal of this application is to elucidate and codify the principles that govern enzyme activation of these reactions.